We propose to apply the tools of the organic chemist as well as the techniques of the enzymologist to the study of the mechanism of action of creatine kinase, an enzyme which uses creatine, phosphocreatine, ADP and ATP as substrates. Creatine kinase serves a dual role as an object for screening both creatine analogs and nucleoside triphosphate analogs of potentially great interest in other biological systems. Our general approach initially involves probing the active site of the enzyme with synthetic structural analogs of the natural substrate to see which functional groups of the substrate molecule allow the analog to act as either a pseudosubstrate or an inhibitor of the normal enzyme catalyzed reaction. Various physical chemical methods, including kinetic studies and nuclear magnetic resonance studies, are proposed in this probing process. The results of these specificity studies will be used in the design of active site-directed irreversible enzyme inhibitors which are analogs of the normal substrates. Where appropriate, the synthesis of a radioactively labeled inhibitor may be desirable in order to facilitate the determination of which side chain group of an amino acid residue of the enzyme has been modified by the irreversible inhibitor.